Oxygen dependency of epidermal growth factor receptor binding and DNA synthesis of rat hepatocytes.
OBJECTIVE
Changes in oxygen availability modulate replicative responses in several cell types, but the effects on hepatocyte replication remain unclear. We have studied the effects of transient nonlethal hypoxia on epidermal growth factor receptor binding and epidermal growth factor-induced DNA synthesis of rat hepatocytes.


METHODS
Lactate dehydrogenase activity in culture supernatant, intracellular adenosine triphosphate content, 125I-epidermal growth factor specific binding, epidermal growth factor receptor protein expression, and 3H-thymidine incorporation were compared between hepatocytes cultured in hypoxia and normoxia.


RESULTS
Hypoxia up to 3 h caused no significant increase in lactate dehydrogenase activity in the culture supernatant, while intracellular adenosine triphosphate content decreased time-dependently and was restored to normoxic levels by reoxygenation (nonlethal hypoxia). Concomitantly, 125I-epidermal growth factor specific binding to hepatocytes decreased time-dependently (to 54.1% of normoxia) and was restored to control levels by reoxygenation, although 125I-insulin specific binding was not affected. The decrease in 125I-epidermal growth factor specific binding was explained by the decrease in the number of available epidermal growth factor receptors (21.37+/-3.08 to 12.16+/-1.42 fmol/10(5) cells), while the dissociation constant of the receptor was not affected. The change in the number of available receptors was not considered to be due to receptor degradation-resynthesis, since immunodetection of the epidermal growth factor receptor revealed that the receptor protein expression did not change during hypoxia and reoxygenation, and since neither actinomycin D nor cycloheximide affected the recovery of 125I-epidermal growth factor binding by reoxygenation. Inhibition of epidermal growth factor-induced DNA synthesis after hypoxia (to 75.4% of normoxia by 3 h hypoxia) paralleled the decrease in 125I-epidermal growth factor binding.


CONCLUSIONS
Transient hypoxia, which caused no increase in lactate dehydrogenase leakage but affected intracellular adenosine triphosphate levels, did, however, modulate the number of available epidermal growth factor receptors without affecting the receptor protein expression, and inhibit the epidermal growth factor-induced DNA synthesis of hepatocytes. This suggests that even transient nonlethal hypoxia affects the epidermal growth factor-induced DNA synthesis of rat hepatocytes through reversible changes in the epidermal growth factor receptor molecule, which depends on oxygen availability.
Hirose
T
T
Department of Gastroenterological Surgery, Kyoto University Graduate School of Medicine, Shogoin, Sakyoku, Japan.
Terajima
H
H
Yamauchi
A
A
Kinoshita
K
K
Furuke
K
K
Gomi
T
T
Kawai
Y
Y
Tsuyuki
S
S
Nakamura
Y
Y
Ikai
I
I
Taniguchi
T
T
Inamoto
T
T
Yamaoka
Y
Y
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D015687:Cell Hypoxia; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis*; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008297:Male; D010100:Oxygen / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar
D007328:Insulin; D004815:Epidermal Growth Factor; D000255:Adenosine Triphosphate; D004247:DNA; D007770:L-Lactate Dehydrogenase; D066246:ErbB Receptors; D010100:Oxygen